ABSTRACT

Like animals and people, insects can serve as both collectors and disseminators of antibiotic resistance genes, as exquisitely demonstrated by a recent study (B. Tian, N. H. Fadhil, J. E. Powell, W. K. Kwong, and N. A. Moran, mBio 3[6]:e00377-12, doi:10.1128/mBio.00377-12, 2012). Notably, the relatively confined ecosystem of the honeybee gut demonstrates a large propensity for harboring a diverse set of tetracycline resistance genes that reveal the environmental burden resulting from the long-time selective pressures of tetracycline use in the honeybee industry. As in humans and animals, these genes have become established in the native, nonpathogenic flora of the insect gut, adding credence to the concept that commensal floras provide large reservoirs of resistance genes that can readily move into pathogenic species. The homology of these tetracycline resistance determinants with those found in tetracycline-resistant bacteria associated with animals and humans strongly suggests a dissemination of similar or identical genes through shared ecosystems. The emergence of linked coresistances (ampicillin and tetracycline) following single-antibiotic therapy mirrors reports from other studies, namely, that long-term, single-agent therapy will result in resistance to multiple drugs. These results contrast with the marked absence of diverse, single- and multiple-drug resistance genes in wild and domestic bees that are not subjected to such selective pressures. Prospective studies that simultaneously track both resistance genes and antibiotic residues will go far in resolving some of the nagging questions that cloud our understanding of antibiotic resistance dissemination.

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.

Commentary

The current global crisis of untreatable multidrug-resistant pathogens has long been associated with the imprudent use of antimicrobials in multiple ecosystems: hospitals, communities, agriculture, horticulture, and aquaculture. The finding of resistance in domestic honeybees reveals yet one more environment that demonstrates the profound ramifications of long-term antibiotic use. The study by Tian et al. (1) on the microenvironment of the honeybee gut provides a particularly exquisite and detailed example of the evolutionary diversity incurred by the long-term use of a single antibiotic, tetracycline, within this industry. The origins of the resistance genes themselves are obscure but are presumed to derive in part from tetracycline use in other ecosystems. For instance, tetracyclines are commonly used as “growth promotants” in animal husbandry (2). So it is no surprise that tetracycline resistance has emerged among food animals, and meaningfully, the determinants for tetracycline resistance are frequent.

The Tian et al. study reveals the profound effects of tetracycline use in beekeeping on the normal gut flora of the honeybee, effects that can potentially have far-reaching impacts on the proliferation and dissemination of resistance genes in the greater environment. It is not often that one finds defined ecosystems which allow, by their very nature, comparisons among different, relatively confined populations. In their examination of the enteric flora of honeybees, the authors describe such a system. The data show a correlation between antibiotic usage, namely, oxytetracycline, to prevent bacterial infections in beehives, and the high frequency of determinants for resistance to tetracycline among the honeybee intestinal flora. What is striking is the variety of tetracycline resistance genes (eight different ones) that are identified in this small ecosystem. While the bee flora is largely Gram negative, the determinants found are associated with both Gram-positive and Gram-negative bacteria, suggesting a nonindigenous origin. While heterogeneous, these tetracycline resistance genes are highly similar, genetically, to those found in bacteria associated with people and farm animals. In contrast, in Europe, little tetracycline is used on honeybee hives, and the bees do not harbor high frequencies of tetracycline-resistant organisms, nor do bumblebees sampled in the wild.

It is interesting to note that although the enteric bee flora contains many different tetracycline-resistant strains, the beehives that Tian et al. (1) studied were not obviously diseased and apparently did not harbor foulbrood pathogens. Thus, in this study group, tetracycline resistance did not thwart treatment, since tetracyclines were not being used against disease. Instead, drug use supported and encouraged the propagation of non-disease-causing bacteria bearing resistance genes. The bacterial strains themselves are normal constituents of the bee gut flora, but the diversity of tetracycline-resistant strains suggests that the resistance genes that they carry were recruited from other environmental sites, including other bees and beehives. The detection of resistance genes among noninfectious strains fits with the conclusion drawn by the Reservoirs of Antibiotic Resistance (ROAR) project of the Alliance for the Prudent Use of Antibiotics (http://www.apua.org), namely, that the vast majority of antibiotic resistance determinants reside not in clinical isolates but in non-disease-causing strains (commensals) (3). In fact, a high frequency of tetracycline resistance is already reported among the native fecal floras of animals and people (2, 4).

One might wonder if the propagation of tetracycline-resistant commensals by tetracycline exposure protects bees from colonization by resistant, disease-causing bacteria. There may not be an easily available niche for entry of disease agents, since the entire ecosystem is colonized with tetracycline-resistant competitors.

The density of the beehive population and the bee intestinal flora potentiates the exchange of resistance genes by two modes of transfer: the resistance genes, which can move between bacterial hosts via mobile genetic elements, and the bees, which can pick up and physically transport tetracycline-resistant bacteria among themselves. Our own studies, performed on a farm, showed that Escherichia coli organisms bearing antimicrobial-resistance genes were carried on flies from farm animals to other animals (5). As both a collector and potential disseminator of resistance genes, the honeybee (and the fly) is a kind of barometer for the spread of resistance determinants among different environments. While no disease was described in the Tian et al. study (1), potential movement of resistance genes to bacterial pathogens in honeybees is always a threat and in fact was documented in foulbrood pathogens beginning in 1996 (6).

Neither we nor Tian et al. assayed for the antibiotic itself, but such studies could have identified the drug’s presence, location, and concentration in the honeybee environs and could also have helped to determine whether a correlation exists between the presence of the antibiotic and the resistant flora. The Tian et al. study presents a compelling picture of a gut flora that is rich in tetracycline resistance genes selected by tetracycline and carried by bees as they visit flowers and interact with the hive.

Do the tetracycline-resistant bacteria exist as a permanent part of this gut ecosystem, or are they only transiently present? In the case of honeybees, it appears that the resistant bee flora is constantly replenished and has consequently established a stable tetracycline-resistant ecosystem. Studies of human volunteers reveal a similar pattern. In his 1988 study of human subjects, Denis Corpet compared the gut bacteria of antibiotic-free volunteers who consumed either normal or only cooked food (7). Corpet recorded a dramatic decrease in tetracycline resistance frequency in volunteers placed on diets of only cooked food, a finding that suggests that uncooked foods provide a steady source of tetracycline-resistant bacteria. Likewise, contact with tetracycline and tetracycline-resistant bacteria helps honeybees maintain their tetracycline-resistant flora.

The authors’ retrospective study looks at long-term antibiotic exposure versus no treatment. It would also have been quite illustrative to conduct a prospective study that examined the timely acquisition of the different resistance determinants. Such a study could help to define their environmental origins, the participating microbial hosts, and the timing and means by which they eventually become stable members of the gut flora. Certainly, the resistance genes associated with the bees are part of a larger ecosystem of resistance genes. The fact that they are highly homologous with the tetracycline resistance genes identified in clinical isolates and among animals suggests that this exchange of resistant determinants is recent and reflects an ongoing process.

Despite the fact that penicillin was never used on these hives, Tian et al. (1) detected ampicillin resistance among the honeybee gut flora. This seeming paradox is reminiscent of prior studies that show that prolonged use of a single antibiotic can lead to multidrug resistance. The genetic basis of this phenomenon resides on plasmids and transposons. In studies of chickens on a farm, 3 months of oxytetracycline use in animal feed led to the appearance of bacteria resistant not only to tetracyclines but also to penicillins and other antibiotics (8). Similarly, single-drug resistance leads to multidrug-resistant selection in farm dwellers (8). The same phenomenon was reported in people taking antibiotics for prolonged periods of time, as for acne, where susceptible skin flora will emerge with resistance, initially to the antibiotic used and later to other antibiotics as well (9). The emergence of resistance to multiple drugs after the application of a single drug is an important concept in understanding gene spread.

All this prompts us to view the many antibiotic-exposed environments as ecosystems that are undergoing processes of gene acquisition and dissemination. Clearly, the comparison here of Europe and the United States is telling. In Europe, honeybee-producing farms avoid antibiotic use for their bees because it tends to be ineffective, can lead to resistance, and favors weak, disease-prone hives. Thus, some bee keepers prefer to destroy infected hives because of the unintended consequences of antibiotic treatment for the ecosystem and environment (6, 10). In fruit orchard husbandry, prophylactic spraying of antibiotics may be helpful in thwarting disease but also carries the consequence of spreading antibiotic resistance, either through selection by drug residues or through gene transfer (10), as described in this study of bees. We have learned that antibiotic use in any environment will lead to changes in the floras harbored by animals, insects, and people sharing that environment. It may be small, as with honeybees, or large-scale, as seen in food animal production.

Tetracyclines are commonly used in animals and in people and are easily shed in the environment as residues. Thus, it may not be so surprising that use of oxytetracycline in honeybees would select for all these different determinants, which themselves could have been preselected by tetracycline use in other nearby environments. The findings raise several questions: why are some tetracycline resistance determinants more commonly found than others? Do the resistance determinants provide an advantage to the growth and survival of the bee existing in a “sea” of tetracycline-resistant bacteria? Why did it take 45 years of oxytetracycline application before disease-causing bacteria emerged with tetracycline resistance? Despite many years of investigation, these and other pressing questions have still eluded definitive answers, and studies directed at these issues will go far in providing a deeper understanding of the complex nature of antibiotic resistance spread and how it can be more successfully contained.